Dr. R K Patel
Artificial Insemination (AI) is the technique by which semen is collected from the male and introduced into the female reproductive tract at proper time with the help of instruments. This has been found to result in a normal offspring. The introduction of semen into the female reproductive tract could be possible by using diluted fresh or frozen semen under most hygienic conditions. The regular testing of frozen semen produced by the bull stations or the semen production unit is mandate mostly by the Government agencies or committees worldwide. These agencies / committees supervise or monitor the semen quality in order to avoid spreading of infectious diseases and ensure the supply of disease free semen for animal breeding programmes. The standards for semen production and distribution are usually based on regulatory programmes to ensure that important diseases are identified and appropriate tests are applied to breeding bulls entering and residing in semen production units. Monitoring committees/ agencies emphasize testing programmes need to be continually improved and updated based on new scientific developments in order to monitor emerging new infectious diseases or pathogens (Truyen et al, 1995). At the international level guidelines are published and revised by the Office International des Epizooties (OIE), the world organization for animal health which was created in 1924 by 28 countries presently located in France. The article focuses on information on infectious agents known to be transmitted by the bull semen, causing infectious diseases and their prevention.
Infectious Bovine Rhinotracheitis (IBR) is a highly contagious, infectious disease that is caused by DNA Bovine Herpesvirus-1 (BHV-1). In addition to causing respiratory disease, this virus can cause conjunctivitis, abortions, encephalitis, and generalized systemic infections. IBR was originally recognized during the early 1950s in feeder cattle in the western US. The IBR virus can persist in clinically recovered animals for years. The virus remains inactive until the animal is placed under stress. The virus is shed in secretions from the eye, nose and reproductive organs. The clinical diseases caused by the virus can be grouped into: 1) respiratory tract infections 2) eye infections 3) abortions 4) genital infections 5) brain infections 6) generalized infections of newborn calves. Bull plays an important role in the dissemination of the disease because the virus is excreted in semen (Van der, 1995). IBR has existed in India since 1976 (Mehrotra, 1977). IBR has been reported from the states of Orissa (Mishra and Mishra, 1987), Karnataka (Mohan et al., 1997) and Gujarat (Singh et al., 1986), and moreover widespread serological evidence of respiratory as well as genital tract infections is reported from most of the states in India (Samal et al., 1980; Suribabu et al., 1984). Although clinical findings of respiratory disease, abortion or Infectious Pustular Vulvovaginitis (IPV) may be highly suggestive of IBR, there is no definitive clinical diagnosis for IBR. Methods of BHV-1 detection currently used by diagnostic veterinary laboratories include virus isolation, examination of tissues by the fluorescent antibody (FA) technique and serological testing; serum neutralization (SN) test or enzyme-linked immunosorbent assay (ELISA). Another method is the ‘Cornell semen test’, in which pooled samples of semen are inoculated into susceptible calves or sheep which are then monitored for neutralising antibodies to BHV-1 (Schultz et al., 1982). The traditional method for detection of BHV-1 in bovine semen is virus isolation and identification in cultures of cells of bovine origin (Straub, 1990). IBR is not a zoonotic disease. No treatment for the virus itself, but supportive care should be provided. Antibiotics in the feed and water are used to treat secondary bacterial infections.
By the year 1997-98, BAIF had developed a specific field diagnostic kit for identifying infected animals. At present, inactivated IBR vaccine (Ibrivax) is available in the country for preventive mass vaccination. The vaccine is developed indigenously from an India strain of BHV-1 and is widely studied on India bovine population. Even both seropositive and seronegative female animals can be vaccinated with Ibrivax. Intervet India (Ex-BAIF) Laboratories, based in Pune is well known for its contribution in the promotion and development of animal health care products. It had launched Ibrivax-IBR oil adjuvant vaccine developed by BAIF Development Research Foundation for the prevention and control of viral Infectious Bovine Rhinotracheitis (IBR) disease. All the breeding bulls should be monitored for virus excretion in their semen before using for the AI.
Bovine Virus Diarrhoea is a viral disease of cattle and other ruminants that is caused by the bovine viral diarrhea virus (BVDV) that is world-wide distributed. However, some countries have recently eradicated the virus. It causes a variety of clinical outcomes that range from the unapparent (sub-clinical) to the more severe (clinical manifestation) including abortion, infertility, an immuno-suppression that underlies calf respiratory and enteric diseases, and most dramatically, the fatal mucosal disease (Baker, 1995). Infections of the breeding female may result in conception failure or embryonic and fetal infection, which results in abortions, stillbirths, teratogenic abnormalities or the birth of persistently infected (PI) calves. PI animals shed BVDV in their excretions and secretions throughout life and are the primary route of transmission of the virus. These animals can usually be readily detected by virus or viral antigen detection assays (RT-PCR, ELISA), except in the immediate post-natal period where colostral antibodies may mask virus presence (Lanyon, 2014). The virus can be detected by using molecular techniques (Hoffmann et al., 2006). BVDV is an RNA virus and the disease is not zoonotic in nature. The prevalence of BVDV antibodies in Indian cattle was 15.29% (50/327) in 16 states compared to 23.21% (26/112) in buffalo in 9 states, with an overall prevalence of 17.31% (76/439) in 17 states (Sudharshana et al., 1999; Mishra et al., 2007). Treatment of BVD is limited primarily to supportive therapy. Once identified, infected animals should be culled. Strategic vaccination and high-quality colostrum could also decrease the proportion of susceptible cattle.
BVDV vaccines are used primarily for disease control purposes. BVDV eradication programmes are being undertaken in many countries. To prevent the generation, PI animals are removed and remaining cattle are vaccinated. Controlling BVDV infections by the vaccine can be challenging due to antigenic variability of the virus and the occurrence of persistent infections. On-going maintenance of the virus in nature is predominantly sustained by PI animals. Traditionally, BVD vaccines fall into two classes: modified live virus or inactivated vaccines. Bovidec is the tried and trusted BVD vaccine, and has a long track record of BVD control in both beef and dairy herds throughout the UK and Ireland. Similarly, Bovilis BVD, an inactivated vaccine containing cytopathogenic BVD virus strain C86 producedby the MSD Animal Health, New Zealand. However, BVD vaccine is not available in India.
Bovine Brucellosis is a contagious disease of livestock with significant economic impact. Brucellosis causes economic losses to the tune of Rs 350 million/year in India (PD-ADMAS, 2012). It is caused by various bacteria of the family Brucella but mainly by Brucella abortus (cattle). In cattle, the disease is characterised by abortion and is often associated with retained placenta, metritis and a subsequent period of infertility. It affects many animal species and humans. It is zoonotic and affecting 5% population of livestock worldwide. The highest incidence is observed in the Middle East, the Mediterranean region, sub-Saharan Africa, China, India, Peru, and Mexico (Chitupil et al., 2015; Mangen et al., 2002). Currently, countries in central and southwest Asia are seeing the greatest increase in cases. A serological survey of brucellosis was performed in 23 states of India. A total of 30,437 bovine sample was screened with Rose Bengal plate test (RBPT) and standard tube agglutination test (STAT) which reveals 1.9% prevalence in cattle and 1.8% in buffaloes (Isloor et al 1998). The Project Directorate on Animal Disease Monitoring and Surveillance (PD-ADMAS) conducted long-term serological studies which indicated 5% of cattle and 3% of buffaloes are infected with brucellosis (Renukaradhya et al., 2002), which was higher than previous studies. In one of his presentations, Singh (2007) observed prevalence rate of brucellosis was 8.58% in cattle from Rajasthan and Bihar states. A number of reports on brucellosis in India reveal a high incidence of the diseases (Aulakh et al., 2008; Jagapur et al., 2013; Shome et al., 2014).
All abortions in cattle in late gestation, starting from the fifth month, should be treated as suspected brucellosis and should be investigated and confirmed clinically. The diagnostic tests are applied with different goals, such as national screening, confirmatory diagnosis, certification, and international trade. Unequivocal diagnosis of Brucella infections can be made only by the isolation and identification of Brucella, but in situations where the bacteriological examination is not practicable, diagnosis must be based on serological methods. There is no single test by which a bacterium can be identified as Brucella. A combination of growth characteristics, serological, bacteriological and/or molecular methods is usually needed. Brucella is nonmotile and smooth. Brucella is Gram negative and usually does not show bipolar staining. Following method for identification of agent:
Stamp staining (modified method of Zeihi-Neelson method) is still often used, even though this technique is not specific to other abortive agents. It provides valuable information for the analysis of aborted material (Alton et al, 1988). This is the usual procedure for the examination of smears of organs or biological fluids that have been previously fixed with heat or ethanol, and by this method, Brucella organisms stain red against a blue background.
Direct isolation and culture of Brucella are usually performed on solid media, developing colonies to be isolated and recognised clearly. A wide range of commercial dehydrated basal media is available, e.g. Brucella medium base, tryptose (or trypticase)–soy agar (TSA). The addition of 2–5% bovine or equine serum is necessary for the growth of strains. On suitable solid media, Brucella colonies are visible after a 2–3-day incubation period. For the diagnosis of animal brucellosis by cultural examination, the choice of samples usually depends on the clinical signs observed. The most valuable samples include aborted fetuses (stomach contents, spleen and lung), fetal membranes, vaginal secretions (swabs), milk, semen and arthritis or hygroma fluids. From animal carcasses, the preferred tissues for culture are head, mammary and genital lymph nodes, spleen, uterus and the udder.
Identification of Brucella organisms can be carried out by a combination of the following tests: organism morphology and Gram or Stamp’s staining, colonial morphology, growth characteristics, urease, oxidase and catalase tests, and the slide agglutination test with an anti-Brucella polyclonal serum. Species and biovar identification requires elaborate tests; phage lysis and agglutination with A-, M- or R-specific antisera. The serum samples can be analysed by Rose Bengal Plate Test (RBPT) according to standard protocol (Alton et al. 1988). The Rose Bengal test (RBT) is a simple, rapid slide-type agglutination assay performed with a stained B. abortus suspension at pH 3.6–3.7 and plain serum. Owing to its apparent simplicity of reading, however, interpretations of the RBT results can be affected by personal experience (Cho et al., 2010).
The advent of molecular techniques allowing identification of Brucella has been developed (Bricker et al.2003) and are in use in certain diagnostic laboratories. Several PCR based methods have been developed. The best validated methods are based on the detection of specific sequences of Brucella spp., such as the 16S-23S genes, the IS711 insertion sequence or the bcsp31 gene encoding a 31-kDa protein (Baddour et al., 2008; Ouahrani-Bettache et al., 1996).
The reactivity of positive samples should be confirmed by the complement fixation test or by enzyme-linked immunosorbent assay (ELISA), both of which can also be used for both screening and confirmation.
The most widely used vaccine for the prevention of brucellosis in cattle is the Brucella abortus S19 vaccine. It is used as a live vaccine and is normally given to female calves between 3 and 6 months of age as a single subcutaneous dose at a concentration of 5-8 × 1010 viable organisms. Whereas, a reduced dose (@ cons. of 3×108 to 3×109 organisms) can be administered subcutaneously to adult cattle. Brucella abortus S19 vaccine induces good immunity to moderate challenge by virulent organisms. Since 1996, B. abortus strain RB51 vaccine has been used for prevention of brucellosis in cattle in some countries but each country is using different concentration of viable strain. Brucella Melitensis and Brucella Abortus/strain 19 are produced by many companies in India.
Leptospirosis is an economically important zoonotic bacterial infection of livestock that causes abortions, stillbirths, infertility, and loss of milk production when chronic. Leptospirosis is worldwide distribution and occurs in man, cattle, buffaloes, pig, sheep, goat, dog, horse, etc. The epidemic presents an increasing incidence in both developing and developed countries (Meites et al., 2004). It manifests as an acute or chronic disease or as a clinical inapparent contagious disease of domesticated and wild animals as well as man. It is caused by spirochetes belonging to genus Leptospira. Leptospira are ubiquitous spirochetes and are spiral shaped bacteria and posses a Gram negative like cell envelope consisting of cytoplasmic and outer membranes. In 2007 meeting of the Subcommittee on the Taxonomy of Leptospiraceae held in Ecuador, some of the previously described genomospecies were given the status of species resulting in a family comprising 13 pathogenic Leptospira species with more than 260 serovars and 6 saprophytic species comprising more than 60 serovars. Serotype hardjo, the predominant in most cattle populations (Bokhout et al., 1989), has been divided into two genotypes: hardjo-prajitno and hardjo-bovis (Thiermann and Ellis, 1986). Persistent infection of the male and female genital tract is also a prominent feature of serovar Hardjo infections. It is expected that additional new species exist and will add to this ever expanding taxa (Adler and Moctezuma, 2010). Survival of the leptospirae depends on the variation in soil and water conditions in the contaminated area. They are susceptible to drying, pH lower than 6 or greater than 8, ambient temperatures lower than 7ºC or higher than 34ºC. They can survive for as long as 183 days in water saturated soil, but survives for less than 30 minutes when soil is air dried. It can survive for very long periods in free surface water (WHO, 2006). Carrier animals, domestic or wild, maintain and propagate leptospires within the population. Infected animals may excrete leptospires intermittently or regularly for months or years, or even for their lifetime. The leptospires dwell in the renal tubules of their animal host. Vaccinated animals may still shed infectious organisms in the urine. Main transmission among animals can be sexual contact or by suckling milk from infected mother.
Leptospirosis in Indian cattle is from the early 20th century. Most outbreaks of leptospirosis in India are reported from the coastal regions of the states of Gujarat, Maharashtra, West Bengal, Orissa, Kerala, Tamil Nadu, Karnataka and the Andaman Islands. Highest rates occur during October to November which coincides with the monsoon season in these parts. A significant outbreaks of leptospirosis have been occurring in the past few years in different parts of India; Orissa (Faine, 1994; Sehgal et al., 2001), Mumbai (Karande et al., 2002) and the Andaman archipelago (Sehgal et al., 1995; Singh et al., 1999), Karnataka (http://www.gideononline.com). Reports from the Southern part of Gujarat revealed 130 deaths within a two month period because of leptospirosis. Many deaths due to leptospirosis were reported from Kochi (Kerala), Surat and Valsad (Gujarat) (Patel et al., 2014; Himani et al., 2013). Most leptospiral infections are subclinical and infection is more common than clinical disease. In India, Adinarayanan et al. (1960) were the first to report leptospirosis among buffaloes in Uttar Pradesh. Subsequently, the prevalence of the disease in animals from various parts of the country has been reported by Rajasekhar and Nanjiah (1971), Srivastava et al. (1983, 1991), Varma et al. (2001), Piramanayagan et al. (2002) and Sivaseelan et al. (2003). Consequent to an outbreak of bovine leptospirosis in Chennai, serological evidence of leptospirosis was evident among human subjects (Ratnam et al., 1983). Prevalence studies being carried out by Indian Veterinary Research Institute during the last 35 years have showed an overall prevalence of 10.1% during 1975-90. During 1991-2000 the overall sero-positivity marginally increased.
Diagnosis of leptospirosis in cattle is relatively straightforward. In general, infected animals develop high titers to the infecting serovar; an antibody titre >1:800 at the time of abortion is considered evidence of leptospirosis. Leptospires can be demonstrated in the placenta and the fetus in some cases by immunofluorescence, PCR, and immunohistochemistry. Diagnosis of serovar Hardjo infection is more difficult and requires a combination of approaches. Serology alone often fails to identify animals infected with serovar Hardjo, because seronegative shedders are common in infected cattle herds. The recommended diagnostic testing strategy includes the primary use of a test (immunofluorescence or PCR) to detect the organism in the urine from a sample of cattle in the herd followed by serologic testing to provide insight into the likely infecting serovar of Leptospira. Cattle with acute leptospirosis can be treated with the label dosage of tetracycline, Oxytetracycline, penicillin, ceftiofur, tilmicosin, or tulathromycin. Leptospires are also highly susceptible to erythromycin, tiamulin, and tylosin, although these antibiotics cannot be relied on to remove the renal carrier state. Injectable, long-acting Oxytetracycline (20 mg/kg) and sustained-release ceftiofur have been shown to effectively eliminate shedding in cattle infected with serovar Hardjo. Vaccination can be combined with antibiotic treatment in the face of an outbreak of leptospirosis, but vaccination alone will not reduce urinary shedding. Leptospirosis in domestic animals can be controlled through vaccination with inactivated whole cells or an outer membrane preparation (Palaniappan et al., 2002). However, vaccine for leptospirosis is not available in India and infected animals are usually treated with antibiotics to eradicate the disease.
Bovine Genital Campylobacteriosis (BGC) is a venereally transmitted bacterial disease caused by Campylobacter fetus subsp venerealis. It is a gram-negative, non-spore forming bacterium, which is microaerophilic (oxygen dependent), fragile, and survives for only 6 hours under normal atmospheric conditions. The disease is subclinical nature in bulls with no overt clinical signs, but they are carriers and can infect females at service (Hum, 1994). The disease is characterised by temporary infertility of female cattle as a result of a subacute diffuse mucopurulent (composed of mucus and pus) cervicitis (inflammation of the cervix), endometritis (Inflammation and infection of the endometrium (lining of the uterus) and salpingitis (inflammation of a Fallopian tube). Abortion occurs in a small percentage of infected cows, months after initial infection (Clark, 1997; Hoerlein, 1981). C fetus subsp fetus is similar to C fetus subsp venerealis that affects cattle and other species; sheep, birds. Although it has been known to cause sporadic abortions in cattle, it is not usually associated with infertility (Eaglesome and Garcia 1992). No disease reported in India, however, it is present in other countries (OIE reports). A definitive diagnosis of genital vibriosis can be difficult and the results of laboratory tests are often disappointing (Andrews and Frank, 1974). Four laboratory tests – serum agglutination, cervical mucus agglutination, fluorescent antibody and culture have been used extensively in the past; however, each of these tests has a number of limitations. For laboratory diagnosis the bacteria may be isolated and identified from preputial scrapings and semen from bulls, and cervico vaginal mucus and aborted fetuses, placentas from females. Infected bulls produce IgA in their preputial secretions. The multiplex PCR described by Hum et al. (1997) is currently the most cited PCR. It enables the amplification of a C. fetus-specific DNA fragment (approximately 200 bp smaller than the 960 bp described in the original publication), as well as a C. fetus subsp. venerealis-specific fragment. Thus, the performance of this multiplex PCR allows differentiation of the two subspecies (C. fetus = one amplification product vs C. fetus subsp. venerealis = two amplification products). Vaccination should start as soon as genital campylobacteriosis is diagnosed. Infected cows and cows at risk should be vaccinated. However, vaccine for BGC is not produced in India.
Trichomoniasis is a human and bovine sexually transmitted disease. Human trichomoniasis is caused by Trichomonas vaginalis and bovine trichomoniasis is caused by Tritrichmonas foetus. Bovine trichomoniasis has resulted in reproductive failure. Fetal loss occurs most often late in the first trimester or early in the second trimester, although late term abortions are also seen. Since this is the only known animal to have a naturally occurring sexually transmitted disease due to trichomonads, it serves as a model of human trichomoniasis. Although the parasite can survive in diluted semen and through the freezing process, the probability of transmitting infection through AI is not known. The human infection is also associated with adverse outcome of pregnancy, including preterm birth, premature rupture of the membranes and low birth weight infants (Cotch et al., 1997; Minkoff et al., 1984; Petrin et al., 1998; Schwelke, 2002). Trichomonosis occurs world-wide, particularly among range cattle (Pefanis et al., 1988; BonDurant et al., 1990; Riley et al., 1995). There are no reports of trichomonosis available in Indian bovine as De et al (1982) did not find infected animals among 13 well-managed herds in West Bengal, India. Only one cow from a rural herd was diagnosed as being infected.
Since these organisms tend to be present only in small numbers, a vigorous scraping of the preputial epithelium is recommended. A diagnostic kit has been developed which consists of a clear plastic pouch with two chambers of selective media. This is inoculated on-site with the sample and is then used for both transport and culture (Thomas et al., 1990). The similar kit is also available with Tamil Nadu Veterinary and Animal Science University (TNVAS), Chennai (Hand book on the detection of trichomonosis feotus under laboratory conditions from field semen or preputial samples). The same culture may be used for DNA extraction and PCR detection of protozoan parasite. The inoculated culture media are examined microscopically for motile trichomonads for up to seven to ten days (Kimsey, 1986). Other ELISA has been developed (BonDurant, 1997; Gault et al., 1995). Molecular-based techniques that use PCR technology have been developed for the identification of T. foetus (Campero et al., 2003; Cobo et al., 2007; Parker et al., 2001). A PCR diagnostic test offers a number of potential advantages, including increased analytical sensitivity, faster diagnostic turnaround time, and the fact that the organisms in the collected sample are not required being viable.
When practical, artificial insemination is an excellent way to prevent or control genital diseases. It has been suggested that artificial insemination should continue until all the cows in a herd have been through at least two pregnancies. The vaccine protects against various bovine infections may not only protect the elite herd, but it is useful to protect against human sexually transmitted diseases. The testing of bulls entering AI should be mandatory.
Further reading: Patel, R K. (2016). Bovine semen transmittable diseases. Blue Cross Book. 34: 11-20.